Figure 5

Complementation to assay the resistance of the Δ ygfZ strain toward plumbagin after expressing homologous constructs. (A) Amino-acid-sequence alignment of E. coli YgfZ (ref|NP_417374), K. pneumoniae YgfZ (Kp_YgfZ; ref|BAH65109), and M. tuberculosis Rv0811c (ref|NP_215326). Residues conserved in all three sequences are marked in black whereas those semi-conserved are boxed in gray; labeled above the alignment are residue numbers of the longest Rv0811c sequence and exceptions are those italicized for which represent the YgfZ residues in E. coli and K. pneumoniae. The cysteine residue in the conserved fingerprint region [23] is asterisked. Inset: amino acid identity between pairs of the three proteins as calculated by Vector NTI (InforMax). (B) Comparison of the activities of different YgfZ constructs to support the growth of the ΔygfZ E. coli strain in the presence of plumbagin. Plasmids were separately transformed into the ΔygfZ strain and assayed for the diameters of the growth inhibition zone as in Figure 1B. Inset: the plasmid-encoded proteins expressed in the transformants were detected by Western blotting using anti-His_x6 antibody; Dank was detected in parallel, to assure a comparable protein loading. Note: pQE60 served as a negative control. NS: no significance; * p < 0.05